The Role Of Notch Signaling In The Feather Branching

Epithelial appendage morphogenesis involves a series of fate determination processes:homogeneous epithelial sheath invaginate into mesenchymal and eventually develop into a sophisticated structure,such as hair,nails,teeth and so on.The feather of birds is a perfect model to study the biological process.The Notch/Delta receptor/ligand pair as an evolutionarily conserved signaling system play a key role in numerous cell fate decisions.Researches have shown that Notch/Delta/Serrate gene are expressed dynamically in feather,which indicates they involve the process of feather branching.But the how Notch/Serrate act out in this system is still unclear.In the article,we used reverse genetics and other modern biological methods to explore the mechanism of Notch signaling in the feather morphogenesis.We first confirmed high expression of Notch1,Serrate1 and Serrate2 in the feather tube by in situ hybridization.And from the results of in situ hybridization,we found 1)in the stagel(S1),which just before the feather branching,Notchl is expressed mainly in the feather epithelium with no graded distribution in the anterior-posterior axis.The expression is much weaker in the basal layer.The expression pattern of Serrate 1 is similar to that of Notch1,with lower expression in the basal keratinocytes,and weaker expression in the most differentiated cells.Strikingly,Serrate2 expression shows a complementary pattern,which is strongly expressed in basal keratinocytes.In the stage3(S3)or the early stages of feather branching,Serratel and Notchl mainly localize to the barb plates while Serrate2 localizes to the marginal plates.Thus,Serratel is coexpressed with Notchl in the feather development,while Serrate2 is expressed in the opposing cell types to the Notchl in the feather epidermal,indicating Notchl and Serrate2 are probably to take the classic lateral inhibition mechanism to regulate feathers morphogenesis.To further identify the function of Notch signaling in the feather branching,we performed RNA interference-mediated knockdown of the three gene respectively.Two independent Lentivirus infection assays resulted in ectopic epithelial branching in the rachis.On the other hand_Notchl overexpression in the branching region led to ectopic rachis formation,which suggested that Notch1 inhibited feather branching and promoted the formation of rachis at high concentration.The phenotype of Serrate1 and Serrate2 overexpression is different,so that their function is different.We next identified how Notch signaling regulates feather branching formation.Firstly,the expression patterns of down-stream target genes of Notch,such as L-finge and Heyl,were examined.In situ hybridization showed they both are expressed in marginal plate.Secondly,we cloned a Notch reporter into lentivirus,where GFP expression was driven by a promoter containing 6X CSL binding elements.Infection of this virus in the developing follicles showed GFP expression in marginal plate cells,indicating activation of Notch signaling specifically in this region.And finally,we overexpressed a secretory form of Serrate2(sSer2),which was known to block Notch signal transduction.This resulted in ectopic rachis formation,a phenotype similar to Notchl overexpression.Together,these results suggest that a lateral inhibition mechanism mediated by Notch signaling is required for epithelial branching.